OBJECTIVE This study investigated the effect of two different follicle stimulating hormone (FSH) preparations (diluted/dynamised and diluted) on the in vitro development and steroid production (estradiol, progesterone and testosterone) of isolated porcine preantral follicle after in vitro culture. METHODS Secondary follicles were cultured in Alpha Minimum Essential Medium (α-MEM+) supplemented with grain ethanol (AL - 0.2%, v/v), diluted/dynamised FSH (rFSH 6cH - 0.05 fg/mL) or diluted-only FSH (1.5 ng/mL) for 4 days. Follicle development was evaluated on the basis of follicular growth, morphology and hormone production. RESULTS The percentage of follicular integrity and extrusion were not affected by the treatments after culture. For all treatments, follicular diameter increased significantly from Day 0 to Day 4. On Day 2 of culture, the estradiol production was significantly higher in AL and diluted-only FSH treatments compared with diluted/dynamised FSH. However, diluted/dynamised FSH showed a significantly higher progesterone production on Day 2. Only on Day 4, the testosterone production was higher in the AL than diluted-only FSH treatments, but similar to diluted/dynamised FSH treatment. Except for diluted/dynamised FSH treatment, progesterone production increased (P < 0.05) from Day 2 to Day 4; only for AL treatment, a significant increase of testosterone production was observed during culture. CONCLUSIONS Compared to control the diluted/dynamised FSH addition increased progesterone production but decreased the estradiol production after in vitro culture of isolated porcine preantral follicles. Taken together the results suggest that at least for progesterone production the mechanism of action of diluted/dynamised FSH differs from its vehicle.